Project Summary Transition metal zinc is the second most abundant trace element in human body and it has been extensively exploited in biological systems for enzymatic catalysis, structural stability and signal transduction. To precisely control the levels of this potential toxic metal, designated transport systems have been evolved. Zinc transport is primarily conducted by the ZnT family for efflux and the Zrt-/Irt-like protein (ZIP) family for influx from either extracellular space or intracellular organelles. The fourteen human ZIPs are broadly involved in a variety of physiological and pathological processes, but little is known about the structure and the transport mechanism of the ZIPs, which hinders the development of novel therapies. Our recent progress of solving the first crystal structure of a prokaryotic ZIP has paved a way towards the ultimate goal of this project - a thorough understanding of the metal transport mechanism of the ZIPs. To achieve this goal, we will focus our research on a prototypical bacterial ZIP and the human protein ZIP4, which is exclusively responsible for zinc uptake from dietary food and involved in genetic disease and cancers, in the following three aspects. In Aim1, we will structurally characterize the outward- facing conformation of the ZIPs by using a combination of structural, computational, biochemical and cell biological approaches. This structural information, together with the previously characterized inward-facing conformation, will enable us to establish an alternating access mechanism in metal transport. In Aim2, we will clarify the roles of the binuclear metal center, which was unexpectedly identified in the middle of the transport pathway, in zinc binding and zinc binding facilitated conformational transition during a transport cycle. The knowledge obtained in Aim1 and Aim2 will help us to depict a full transport cycle. In Aim3, we will identify the molecular determinants of substrate specificity in the human ZIPs. We will make comparison between ZIP4 and ZIP8, whose physiological substrates are zinc and manganese, respectively, and identify the key residues/elements dictating the substrate preference by systematic mutagenesis and activity measurement. Overall, the findings obtained through these efforts will greatly increase knowledge of the unique metal transport mechanism of the ZIPs, which will fill the knowledge gap in zinc signaling and zinc homeostasis, expand the structural and mechanistic diversity of membrane transporters, and importantly, facilitate the development of new therapeutics against human diseases, including several types of cancers.